Cytokine 74 (2015) 327–330

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Cytokine
journal homepage: www.journals.elsevier.com/cytokine

Short Communication

IL-18 contributes to susceptibility to Leishmania amazonensis infection

by macrophage-independent mechanisms
Louisa M.A. Sousa a,b, Matheus B.H. Carneiro a, Liliane M. dos Santos a, Caio Cotta Natale a,
Magda E. Resende a, David M. Mosser b, Leda Q. Vieira a,⇑
a
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
b
Maryland Pathogen Research Institute, University of Maryland, College Park, MD, USA

a r t i c l e i n f o a b s t r a c t

Article history: We evaluated the role of IL-18 during Leishmania amazonensis infection in C57BL/6 mice, using IL-18KO
Received 27 May 2014 mice. We showed that IL-18 is involved in susceptibility to L. amazonensis, since IL-18KO mice presented
Received in revised form 8 October 2014 reduced lesions and parasite loads. Because macrophages are the host cells of the parasite, we investi-
Accepted 23 January 2015
gated if macrophages were involved in IL-18-mediated susceptibility to L. amazonensis. We showed that
Available online 23 May 2015
macrophages obtained from WT or IL-18KO responded similarly to L. amazonensis infection. Moreover,
we showed that C57BL/6 macrophages do not respond to IL-18, since they do not express IL-18R.
Keywords:
Therefore, macrophages are not involved in IL-18-mediated susceptibility to L. amazonensis.
IL-18
Leishmania amazonensis
Ó 2015 Elsevier Ltd. All rights reserved.
Macrophage
Leishmaniasis

1. Introduction contributes to an increase in Th2 responses [4]. Besides, during

Interleukin-18 (IL-18) is a pleiotropic cytokine secreted by
activated macrophages. Originally, IL-18 was considered a proin-
flammatory cytokine, due to its ability to induce IFN-c production
and T cell and NK cell proliferation [1]. IL-18 acts synergically with
IL-12 activating NK cells and inducing the development of Th1 cells
[2]. However IL-18 is also able to induce Th2 responses. In the
absence of IL-12, IL-18 acts synergically with IL-2 to induce the
expression of IL-13 by NK cells and T cells, and IL-4 and IL-10, by
anti-CD3-stimulated T cells [3].
The role of IL-18 during Leishmania infection varies depending
upon the Leishmania species and the genetic background of the
host. In C57BL/6 mice, which are highly resistant to Leishmania
major infection and produce strong Th1 responses, IL-18 seems
to have a marginal role. IL-18/ C56BL/6 mice presented increased
lesions compared to WT mice at early points of infection, but they
still were able to control and heal the infection. IL-18 contributed
significantly to resistance in 129xC1 and DBA/1 mice, which are
moderately resistant to L. major [4]. On the other hand, during
L. major infection in BALB/c mice, which produce a Th2 response
probably due to the lack of persistent IL-12 stimulation, IL-18
⇑ Corresponding author at: Departamento de Bioquímica e Imunologia, Instituto
de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos
6627, 31270-901 Belo Horizonte, MG, Brazil. Tel.: +55 31 34092656.
E-mail address: lqvieira@icb.ufmg.br (L.Q. Vieira).
Leishmania donovani infection, IL-18 has a protective role [5], while
during Leishmania mexicana infection IL-18 promotes a Th2
response and exacerbation of the infection [6]. Therefore it is clear
that the effect of IL-18 depends upon the cytokine environment
generated during the infection. In this study, our goal was to
investigate the role of IL-18 during L. amazonensis infection.
2. Materials and methods
Six-week-old female C57BL/6 and IL-18KO
(B6.129P2-Il18tm1Aki/J) mice were used. Experimental procedures
were approved and conducted according to the guidelines of the
Animal Research Ethical Committee (CETEA protocol number
050/09), UFMG. L. amazonensis IFLA/BR/67/PH8 was used in this
study. Infective stage metacyclic promastigotes were purified from
stationary cultures by density gradient centrifugation, as described
previously [7]. Mice were inoculated intradermally into the ear
dermis with 1000 metacyclic L. amazonensis parasites. The evolu-
tion of the infection was monitored weekly by measuring the
thickness of the ear lesion with a digital caliper (Starrett, Brazil).
Parasite burden was determined by performing limiting dilution
analysis. Parasite numbers are expressed as the negative log10
dilution at which parasite growth was observed. Arginase activity
was evaluated as previously described [7].
Bone marrow-derived macrophages (BMM/) were prepared as
previously described [7]. A total of 5  105 BMM/ per well were

http://dx.doi.org/10.1016/j.cyto.2015.01.021
1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
328 L.M.A. Sousa et al. / Cytokine 74 (2015) 327–330

plated overnight in a 48-well plate in DMEM/F12. Cells were then
washed and stimulated with: LPS (10 ng/mL), IL-4 (10 ng/mL),
IL-18 (50 ng/mL or 100 ng/mL). Macrophages were infected with
stationary-phase promastigotes of L. amazonensis at parasite to
macrophage ratio of 5:1. Cytokines were measured in culture
supernatants after 24 h stimulation by sandwich ELISA using the
following antibody pairs from BD Pharmingen™ according to man-
ufacturer’s instructions: IL-12p40, C15.6 and C17.8; IL-10,
JESS-2A5 and JES5-16E3. Nitric oxide (NO) production was
estimated from the accumulation of NO 2 in the medium by
Griess reaction after 24 h of stimulation. Quantification of
Leishmania intracellular growth in macrophages was performed
as described in [8].
BMM/ (5  106 cells per reaction) were subjected to RNA
extraction using TRIzol reagent. The contaminating DNA was
removed by RNase-free DNase I treatment. ThermoScript RT-PCR
system (Invitrogen, USA) was used to generate cDNA from RNA
by using oligo(dT)20. Reverse transcription polymerase chain reac-
tion (RT-PCR) was used to measure IL-18 and IL-18R mRNA levels.
For amplification of IL-18, IL-18R and GAPDH, we used the follow-
ing primer pairs: IL-18 sense: 50 -CTGGCTGTGACCCTCTCTGT-30 ,
IL-18 anti-sense: 50 -ATCTTCCTTTTGGCAAGCAA-30 , IL-18R sense:
50 -ATGCCG AGTTTGGAGATGAG-30 , IL-18R anti-sense: 50 -GCTGTCC
TCTTTCCTGATGC-30 , GAPDH sense: 50 -AAGGTCGGTGTGAACGGAT
TT-30 , GAPDH anti-sense: 50 -AATTTGCCGTGAGTGGAGTCATAC-30 .
Amplification products were resolved on 1% SYBRÒ Safe
DNA-stained agarose gels.
For flow cytometry analysis, BMM/ from WT and IL18 KO mice
were incubated with IL-12 (40 ng/mL) + IL-18 (100 ng/mL), LPS
(10 ng/mL), IL-4 (10 ng/mL) or IFN-c (50 U/mL). Spleen cells from
WT mice were also stimulated and used as positive controls.
After 24 h stimulation, cells were washed and stained with the fol-
lowing antibodies: anti-CD11b (clone M1/70), anti-F4/80 (clone
BM8), anti-MHC II (clone AF6-120.1), anti-CD3 (clone 17A2) and
anti-CD128 (clone P3TUNYA) from eBioscience and anti-CD11c
(clone HL3) from BD Pharmingen™. Briefly, 106 cells were incu-
bated for 20 min with the antibodies plus Fc block (BD
Pharmingen™). Cells were then washed twice and resuspended
in PBS with 1% FBS and 0.01% sodium azide. Flow cytometry data
was acquired using a BD FACSCanto II flow cytometer. Analysis
was performed using the software FlowJo X 10.0.7.
3. Results
To investigate the role of IL-18 during L. amazonensis infection,
we infected wild type mice (WT) and IL-18KO mice with 1000
metacyclic forms of L. amazonensis. The course of infection was fol-
lowed for twelve weeks. Infection with low doses of L. amazonensis
produced much smaller lesions in IL-18 KO compared to WT mice
(Fig. 1A). The development of smaller lesions was associated with
reduced parasite loads in the ears and in the draining lymph nodes
at weeks 8 and 12 post-infection (Fig. 1B and C). IL-18KO mice also
presented reduced arginase activity in the lesions compared to WT
mice (Fig. 1D). Surprisingly, no differences in IFN-c or IL-4 by
antigen-stimulated draining lymph node cells were found (data
not shown). Therefore, these results indicate that IL-18 is involved
in susceptibility to L. amazonensis infection.
Because macrophages are host cells for L. amazonensis and since
we found no differences in Th1/Th2 balance, we hypothesized that
IL-18 could be acting on macrophages favoring the survival of the
parasite inside these cells. To test this hypothesis, we infected
BMM/ obtained from WT and IL-18KO mice with L. amazonensis
(MOI 5:1) and treated with LPS for evaluation of IL-10, IL-12 and
NO production as well as arginase activity (data not shown). We
found that WT and IL-18KO mice behaved similarly. We also com-
pared the ability of IL-18 KO and WT macrophages to kill
Leishmania parasites (data not shown). The number of
parasites/100 macrophages and the percentage of infection were
similar between WT and IL-18KO BMM/. Treatment with LPS
and IFN-c successfully activated macrophages to eliminate

Fig. 1. IL-18 KO mice developed reduced lesions, decreased parasite loads and lower levels of arginase activity compared to WT mice. (A) lesion development expressed by
the increase in ear thickness. (B) assessment of parasite burdens in the ears. (C) assessment of parasite burdens in the draining lymph nodes. (D) arginase activity in the
lesions. Data represent mean ± SE from 5 mice/group. Results are representative of at least three independent experiments. ⁄ indicates p < 0.05 (Mann–Whitney test).
 L.M.A. Sousa et al. / Cytokine 74 (2015) 327–330 329

parasites in both groups. These data suggest that the
IL-18-mediated mechanism of susceptibility to L. amazonensis does
not involve the direct effect of IL-18 on macrophages.
Because we found no significant differences between IL-18 KO
and WT macrophages, we investigated if BMM/ could respond to
IL-18. Firstly, we tested the effect of IL-18 on uninfected C57BL/6
BMM/. BMM/ were treated with different doses of IL-18 (from
6.25 to 100 ng/mL). We showed that treatment with IL-18 had no
effect on IL-10, IL-12 or NO production, and on arginase activity
in BMM/ (Fig. 2). Smaller concentrations of IL-18 are not
shown, but they had no effect either. Treatment with IL-18 had
no effect on the differentiation of classically activated
macrophages (CA-M/) or alternatively activated macrophages
(AA-M/) (Fig. 2).

Fig. 2. C57BL/6 bone marrow-derived macrophages (BMM/) do not respond to treatment with IL-18 because they do not express IL-18R. A–D: BMM/ were cultivated for
24 h with different concentrations of IL-18 in absence or presence of LPS or IL-4. (A) levels of IL-10 in supernatants by ELISA. (B) levels of IL-12 by ELISA. (C) arginase activity in
BMM/ monolayers. (D) nitrite levels by Griess reaction. E: IFN-c in culture supernatants of control and IL-12 and IL-18-treated splenocytes, measured by ELISA. Values are
expressed as the means ± SD. ⁄ indicates p < 0.05 between IL-12 alone and IL-12 + IL-18. # indicates p < 0.05 between IL-18 [100 ng/mL] and IL-12 + IL-18 [100 ng/mL] (Mann–
Whitney test). F: Expression of GAPDH, IL-18 and IL-18R genes (by RT-PCR) in BMM/ monolayers. BMM/ were obtained from C57BL/6 mice and were cultivated in the
following conditions: 1ane 1 – untreated; lane 2 – treated with LPS (classically activated M/); lane 3 – treated with LPS and immune complex [OVA + anti-OVA] (regulatory
M/); lane 4 – treated with IL-4 (alternatively activated M/); lane 5 – treated with IL-18 (100 ng/mL); lane 6 – treated with LPS and IL-18 (100 ng/mL). G: Expression of
GAPDH and IL-18R in splenocytes by RT-PCR. Splenocytes were cultivated in the following conditions: lane 1 – untreated, lane 2 – treated with IL-12 and IL-18. H: Expression
of CD128 (IL-18R) on the surface of CD11b+, F4/80+, CD11c BMM/ by flow cytometry. I: Expression of CD128 on the surface of C57BL/6 splenocytes by flow cytometry. Data
are representative of at least two independent experiments with similar results.
330 L.M.A. Sousa et al. / Cytokine 74 (2015) 327–330
IL-18 has a well-described effect on T cells and B cells. It acts
alone or in synergy with IL-12 to induce IFN-c production by these
cells [2]. To make sure that the IL-18 used was biologically active,
we treated splenocytes with IL-18 alone or in combination with
IL-12 for evaluation of IFN-c production. IL-18 alone at
100 ng/mL was able to induce the production of 2 ng/mL of
IFN-c. In combination with IL-12, treatment with IL-18 induced
the production of high levels of IFN-c (Fig. 2E). Therefore, we con-
cluded that the IL-18 we used in our experiments was biologically
active.
Our next goal was to investigate if BMM/ express IL-18 and
IL-18R. We found that resting BMM/, CA-M/, AA-M/ and R-M/
express IL-18 constitutively (Fig. 2F). We also confirmed that LPS
is a potent inducer of IL-18 expression in macrophages. However
macrophages could not respond to IL-18, because they did not
express IL-18R under any of the circumstances studied (Fig. 2F
and H). We demonstrated the absence of IL-18R expression by
RT-PCR (Fig. 2F) and flow cytometry (Fig. 2H). Splenocytes, on
the other hand, expressed constitutively IL-18R (Fig. 2G and I).
We also tested if pre-treatment with IL-12 or IFN-c could induce
the expression of IL-18R in BMM/. BMM/ did not express IL-18R
following IL-12 or IFN-c pre-treatment (data not shown). Besides,
infection with L. amazonensis was not able to induce the expression
IL-18R in resting, CA-M/, AA-M/ and R-M/ (data not shown).
4. Discussion
To our knowledge, we are reporting for the first time that
C57BL/6 BMM/ do not express IL-18 receptor constitutively.
Others have found IL-18 receptor in peritoneal exudates [9].
These resident macrophages do represent a different population
from the BMM/ [10], and those cell preparations were not pure
[9]. The effects of IL-18 have been mainly described on T cells, B
cells and NK cells [3]. Besides IL-18 also acts on dendritic cells,
mast cells and basophils [11]. The effects of IL-18 on macrophages
were suggested in few studies. One study showed that peritoneal
macrophages produced MCP-1 in response to IL-18 in an
IL-12-dependent mechanism [12]. In another study, it was shown
that stimulation with both IL-12 and IL-18 induced the production
of IFN-c by peritoneal macrophages [13]. However, these studies
were performed with macrophages obtained from murine peri-
toneal exudates, which could be contaminated with lymphoid cells
[14]. Even minor contaminations with lymphoid cells were shown
to alter significantly the responses of a pure population of
macrophage-like cells (Raw 264.7) [14], therefore the activation
of macrophages in response to IL-12 and IL-18 might involve the
direct stimulation of residual lymphoid cells to secrete IFN-c.
Opposing this argument, one study showed that BMM/ secreted
IFN-c in response to stimulation with IL-12 and IL-18 [15]. It does
not seem to be the case in our study, since BMM/ obtained from
both WT and IL-18KO mice had similar ability to bear and elimi-
nate parasites when activated with IFN-c (data not shown).
Our results indicate that IL-18 contributes to susceptibility to L.
amazonensis in C57BL/6 mice. However, the mechanism of suscep-
tibility does not involve the direct effect of IL-18 on macrophages,
since we showed that BMM/ obtained from C57BL/6 do not
respond to IL-18, because they do not express its receptor. Since
IL-18 can induce Th2 responses, one could speculate that IL-18
could be contributing to susceptibility by inducing Th2 responses.
However, Th2 responses do not represent an important mechanism
of susceptibility to L. amazonensis in C57BL/6 mice. On the other
hand, CD4+ T cells with characteristics of Th1 cells are thought to
be associated with pathogenesis of L. amazonensis infection in
C57BL/6 mice [16]. Besides, IFN-c can promote L. amazonensis
replication in macrophages in vitro in certain conditions by induc-
tion of the expression of cationic amino acid transporter 2B
(CAT2B) that transports L-arginine, which is used directly by the
parasite for growth [17]. Therefore, we are now investigating if
IL-18 is contributing to susceptibility to L. amazonensis by inducing
pathogenic T cell subsets.
In conclusion, our results indicate that IL-18 contributes to the
susceptibility of C57BL/6 mice to L. amazonensis and the mecha-
nism involved is not mediated by macrophages, since they do
not express IL-18R.
Acknowledgements
LMAS, MBHC, MER and LQV are CNPq fellows. This work was
supported by FAPEMIG Grant Number APQ-01599-11 and NIH
Grant U01 AI088650.
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